Journal: PLoS Neglected Tropical Diseases

Article Title: Identification of MicroRNA-Like RNAs in Mycelial and Yeast Phases of the Thermal Dimorphic Fungus Penicillium marneffei

doi: 10.1371/journal.pntd.0002398

Figure Lengend Snippet: Analysis of total and small RNA sequences in mycelial and yeast phase of P. marneffei.

Article Snippet: The potential targets of milRNA candidates were predicted using the predicted gene sequences, including their 5′ and 3′ UTRs, of the P. marneffei strain PM1 and ATCC strain 18442 draft genomes by the RNAhybrid program with or without mismatches or insertions at positions 9–11 of the milRNA and with parameters that encourage complete complementarity at the seed region (positions 2–7 of the milRNA) .

Techniques:

Journal: PLoS Neglected Tropical Diseases

Article Title: Identification of MicroRNA-Like RNAs in Mycelial and Yeast Phases of the Thermal Dimorphic Fungus Penicillium marneffei

doi: 10.1371/journal.pntd.0002398

Figure Lengend Snippet: Potential milRNA candidates in mycelial and yeast phase of P. marneffei .

Article Snippet: The potential targets of milRNA candidates were predicted using the predicted gene sequences, including their 5′ and 3′ UTRs, of the P. marneffei strain PM1 and ATCC strain 18442 draft genomes by the RNAhybrid program with or without mismatches or insertions at positions 9–11 of the milRNA and with parameters that encourage complete complementarity at the seed region (positions 2–7 of the milRNA) .

Techniques:

Journal: PLoS Neglected Tropical Diseases

Article Title: Identification of MicroRNA-Like RNAs in Mycelial and Yeast Phases of the Thermal Dimorphic Fungus Penicillium marneffei

doi: 10.1371/journal.pntd.0002398

Figure Lengend Snippet: (A) Predicted domains of Dicer and QDE-2 proteins in P. marneffei strain PM1. Black bars represent the full protein sequence. The boxes represent the identified domains, each with its starting and stopping amino acid. Both DCL-1 and DCL-2 of P. marneffei contain a DEAD box, a helicase C domain (hel C), a double stranded RNA binding domain (dsRBD), and two RNase III domains (RNase IIIa and RNase IIIb). QDE-2 contains a PAZ domain ,a Piwi domain and a DUF1785 domains. Phylogenetic tree showing the relationship of predicted protein sequences of (B) dcl-1 , (C) dcl-2 , (D) qde-2 and (E) ITS of P. marneffei to homologues in other fungi constructed by maximum-likelihood method with Homo sapiens (DCL-1,DCL-2 and QDE-2) and Ustilago maydis (ITS) as the root. The thermal dimorphic pathogenic fungi are highlighted. A total of 914, 764 and 525 amino acid positions for dcl-1 , dcl-2 and qde-2 and 465 nucleotide positions for ITS were included in the analysis respectively. Bootstrap values were calculated as percentages from 1000 replicates and only values ≥70% were shown. The scale bars indicate the estimated number of substitutions per 5, 5, 5 amino acids and 10 bases respectively. Names and accession numbers are given as cited in GenBank database.

Article Snippet: The potential targets of milRNA candidates were predicted using the predicted gene sequences, including their 5′ and 3′ UTRs, of the P. marneffei strain PM1 and ATCC strain 18442 draft genomes by the RNAhybrid program with or without mismatches or insertions at positions 9–11 of the milRNA and with parameters that encourage complete complementarity at the seed region (positions 2–7 of the milRNA) .

Techniques: Sequencing, RNA Binding Assay, Construct

Journal: PLoS Neglected Tropical Diseases

Article Title: Identification of MicroRNA-Like RNAs in Mycelial and Yeast Phases of the Thermal Dimorphic Fungus Penicillium marneffei

doi: 10.1371/journal.pntd.0002398

Figure Lengend Snippet: Northern blot analyses of small RNA samples in wild-type (WT), dcl-1 KO , dcl-2 KO , dcl DKO and qde-2 KO strains of P. marneffei showing that the production of milRNA of (A) PM-milR-M1 and (B) PM-milR-M2 requires DCL-2 but not DCL-1 or QDE-2. The ethidium bromide-stained denaturing gel in the bottom panel showed equal loading of RNA. Predicted structures of pre-milRNA of PM-milR-M1 and PM-milR-M2 , with their milRNA and paired milRNA* sequences as labeled in red and green respectively, are shown next to the northern blot analyses. The probe sequences used for northern blot analyses are marked.

Article Snippet: The potential targets of milRNA candidates were predicted using the predicted gene sequences, including their 5′ and 3′ UTRs, of the P. marneffei strain PM1 and ATCC strain 18442 draft genomes by the RNAhybrid program with or without mismatches or insertions at positions 9–11 of the milRNA and with parameters that encourage complete complementarity at the seed region (positions 2–7 of the milRNA) .

Techniques: Northern Blot, Staining, Labeling

Journal: PLoS Neglected Tropical Diseases

Article Title: Identification of MicroRNA-Like RNAs in Mycelial and Yeast Phases of the Thermal Dimorphic Fungus Penicillium marneffei

doi: 10.1371/journal.pntd.0002398

Figure Lengend Snippet: Relative mRNA expression of (A) PM-milR-M1 gene, (B) RanBP10 , (C) benzoate 4-monooxygenase cytochrome P450 and (D) a conserved hypothetical protein in mycelial phase of wild type strain PM1 and knockdown strain PM-milR-M1 KD by qRT-PCR. Results were obtained from three independent experimental replicates.

Article Snippet: The potential targets of milRNA candidates were predicted using the predicted gene sequences, including their 5′ and 3′ UTRs, of the P. marneffei strain PM1 and ATCC strain 18442 draft genomes by the RNAhybrid program with or without mismatches or insertions at positions 9–11 of the milRNA and with parameters that encourage complete complementarity at the seed region (positions 2–7 of the milRNA) .

Techniques: Expressing, Knockdown, Quantitative RT-PCR

Journal: Cell reports

Article Title: Mitochondrial reactive oxygen species regulate acetyl-CoA flux between cytokine production and fatty acid synthesis in effector T cells.

doi: 10.1016/j.celrep.2025.115430

Figure Lengend Snippet: Figure 1. TMEM11 deficiency decreases the effector function of Th1 cells (A) Representative immunoblot showing expression of TMEM11 in effector T cells cultured under non-polarizing (ThN) and Th1-, Th2-, non-pathogenic (np-) Th17-, pathogenic (p-) Th17-, and inducible Treg (Treg)-polarizing conditions. b-actin, loading control. (B) Representative flow plots and line graphs showing cell trace violet (CTV) dilution rate in WT and Tmem11/ T cells cultured under Th1-polarizing conditions for 4 days. Unstimulated WT naive T cells served as control (light gray histogram). (C) Representative flow plots and bar graphs showing IFN-g-expressing populations and mean fluorescence intensity (MFI) of IFN-g in WT and Tmem11/ cells cultured under Th1-polarizing conditions. Cells were restimulated on day 4 with anti-CD3 and anti-CD28 antibodies for 5 h. (D) Representative flow plots (left) and bar graphs showing MFI of TBET expression (right) in WT and Tmem11/ cells cultured under Th1-polarizing conditions. WT T cells cultured under Th2-polarizing conditions were used as negative controls (gray, flow plot). (E) Representative flow plots and bar graphs showing IFN-g and TBET expression in CD4+ cells from the draining lymph nodes of control or Tmem11/ mice injected with MOG peptide for EAE induction and cultured under Th1-expansion conditions for 72 h. (F) Time course of the mean clinical score (left) and body weight measurement (right) of EAE in Rag2/ recipients of control or Tmem11/ draining lymph node cells cultured under Th1-expansion conditions. The line graphs show mean ± SEM from the indicated number of animals from one representative experiment of a total of three experiments. (G) Scatterplots showing the numbers of total mononuclear (left) or CD4+ T cells (right) isolated from the CNS of recipients of control or Tmem11/ cells at the peak of EAE. (H) Representative flow plots showing the cytokine profile of CD4+ T cells from the CNS of recipients of control or Tmem11/ cells at the peak of the disease. Bar graphs show average (±SEM) of normalized frequency of IFN-g+ and GM-CSF+ cells. Individual points in bar graphs in (C), (D), (E), (G), and (H) show data from independent animals. Representative immunoblots and graphs summarize results from at least three independent experiments except where stated otherwise. Data represent means ± SEM; significance was determined by unpaired two-tailed t test (B, C, D, G, and H) and Mann-Whitney U test (F). ###p < 0.0001, *p < 0.05, and **p < 0.005. N.S., not significant.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies TMEM11 Proteintech Cat# 16564-1-AP MIC60 Proteintech Cat# 10179-1-AP MIC10 Bioss antibodies Cat# BS-15029R OXPHOS Abcam Cat# MS604-300 OPA1 BD biosciences Cat# 612606 FLAG tag Millipore Sigma Cat# F3040 b-actin Santa Cruz Biotechnology Cat# sc-47778 anti-CD3 antibody Bio X Cell Cat# BE0001 anti-CD28 antibody Bio X Cell Cat# BE0015 anti-mIFN-g antibody Bio X Cell Cat# BE0055 anti-mIL-4 antibody Bio X Cell Cat# BE0045 anti-hIFN-g Ab-PE (45.B3) ThermoFisher Scientific Cat# 50-111-86 anti-IL-2 Ab-PE (MQ1-17H12) ThermoFisher Scientific Cat# 12-7029-81 anti-TNF Ab-APC (MAb11) ThermoFisher Scientific Cat# 17-7349-41 anti-T-bet Ab-APC (4B10) ThermoFisher Scientific Cat# 2124193 anti-IL-4 Ab-APC (11B11) ThermoFisher Scientific Cat# 17-7041-82 anti-IL-17A Ab-APC (eBio17B7) ThermoFisher Scientific Cat# 17-7177-81 anti-IFN-g Ab-PE (XMG1.2) ThermoFisher Scientific Cat# 12-7311-82 anti-GM-CSF Ab-FITC (MP1-22E9) ThermoFisher Scientific Cat# 11-7331-82 anti-Foxp3 Ab-APC (FJK-16s) ThermoFisher Scientific Cat# 17-5773-82 anti-RORgT Ab-PE (AFKJS-9) ThermoFisher Scientific Cat# 12-6988-82 anti-CD4 Ab-PerCP (RM4-5) ThermoFisher Scientific Cat# 46-0042-82 anti-CD8b Ab-PE (H35-17.2) ThermoFisher Scientific Cat# 12-0083-82 anti-CD44 Ab-PerCP-Cy5.5 (IM7) ThermoFisher Scientific Cat# 45-0441-80 anti-CD62L Ab-APC (MEL-14) ThermoFisher Scientific Cat# 17-0621-81 anti-CD25 Ab-APC (PC61.5) ThermoFisher Scientific Cat# 17-0251-81 anti-CD69 Ab-PerCP-Cy5.5 (H1.2F3) ThermoFisher Scientific Cat# 45-0691-80 anti-CCL3 Ab-eFluor660 (DNT3CC) Invitrogen Cat# 50-7532-82 anti-CCL5 Ab-APC (21445) Invitrogen Cat# MA5-23557 anti-mouse H3K4ac Ab-FITC (EPR16596) Abcam Cat# ab176799 anti-mouse H3K9ac Ab-FITC (C5B11) Cell Signaling Cat# 9649 anti-mouse histone H3 Ab-PE (D1H2) Cell Signaling Cat# 82241S anti-mouse H3K27ac Ab- FITC (D5E4) Cell Signaling Cat# 15485S anti-mouse H3K27me3 Ab (C36B11) Cell Signaling Cat# 9733 anti-mouse H3K4me3 Ab-PE (D1A9) Cell Signaling Cat# 55800S OKT3 BD Cat# 555330 anti-hCD28 antibody Bio X Cell Cat# BE0248 Goat anti-hamster antibody MP Biomedicals Cat# 0855397 anti-Acetyl-Histone H3 (Lys9) antibody (C5B11) Cell Signaling Cat# 9649 Anti-Glucose Transporter GLUT1 antibody Abcam Cat# ab115730 Bacterial and virus strains E. coli DH5a Thermo Fisher Scientific Cat# 18265017 (Continued on next page) Cell Reports 44, 115430, March 25, 2025 15

Techniques: Western Blot, Expressing, Cell Culture, Control, Injection, Isolation, Two Tailed Test, MANN-WHITNEY

Journal: Cell reports

Article Title: Mitochondrial reactive oxygen species regulate acetyl-CoA flux between cytokine production and fatty acid synthesis in effector T cells.

doi: 10.1016/j.celrep.2025.115430

Figure Lengend Snippet: Figure 2. TMEM11 deficiency inhibits selective cytokine production in Th1 cells (A) Volcano plot of differentially expressed genes (DEGs) in control versus Tmem11/ cells cultured under Th1-polarizing conditions and restimulated with anti- CD3 and anti-CD28 antibodies for 5 h. Genes significantly (p adjusted < 0.01) upregulated and downregulated are depicted in red and blue, respectively. (B) Gene set enrichment analysis (GSEA) from control versus Tmem11/ Th1 cells. Phenotype correlation in WT (red) and Tmem11/ cells (blue) is shown. (C) Heatmaps showing Z-score-row-normalized expression values of genes within the indicated gene ontology sets in control versus Tmem11/ Th1 cells. (D) Transcript expression of indicated genes in control and Tmem11/ Th1 cells under resting conditions and 5 h after stimulation with anti-CD3 and anti-CD28 antibodies. Shown are pooled triplicates from two independent experiments. (E) Representative flow plots and bar graphs indicating frequency and MFIs showing expression of CCL3 and CCL5 in control and Tmem11/ Th1 cells. Each dot in the bar graphs represents data obtained from an independent experiment. (F) Transcript analysis of Th1-related transcription factors (top) and pro-inflammatory cytokines (bottom) in the cells from the draining lymph nodes of control or Tmem11/ mice injected with MOG peptide for EAE induction after culturing under Th1-expansion conditions with IL-12 for 72 h. (G) Representative flow plots showing the cytokine profile of CD4+ T cells from the CNS of Rag2/ recipients of control or Tmem11/ cells at the peak of the disease (left). Bar graphs show averages (±SEM) of normalized frequency of cells expressing indicated cytokines (right). All experiments are representative of three biological replicates (unless otherwise indicated) with similar results. RNA-seq data were generated from experiments performed in triplicate. *p < 0.05, **p < 0.005, and ***p < 0.0005 (unpaired two-tailed t test).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies TMEM11 Proteintech Cat# 16564-1-AP MIC60 Proteintech Cat# 10179-1-AP MIC10 Bioss antibodies Cat# BS-15029R OXPHOS Abcam Cat# MS604-300 OPA1 BD biosciences Cat# 612606 FLAG tag Millipore Sigma Cat# F3040 b-actin Santa Cruz Biotechnology Cat# sc-47778 anti-CD3 antibody Bio X Cell Cat# BE0001 anti-CD28 antibody Bio X Cell Cat# BE0015 anti-mIFN-g antibody Bio X Cell Cat# BE0055 anti-mIL-4 antibody Bio X Cell Cat# BE0045 anti-hIFN-g Ab-PE (45.B3) ThermoFisher Scientific Cat# 50-111-86 anti-IL-2 Ab-PE (MQ1-17H12) ThermoFisher Scientific Cat# 12-7029-81 anti-TNF Ab-APC (MAb11) ThermoFisher Scientific Cat# 17-7349-41 anti-T-bet Ab-APC (4B10) ThermoFisher Scientific Cat# 2124193 anti-IL-4 Ab-APC (11B11) ThermoFisher Scientific Cat# 17-7041-82 anti-IL-17A Ab-APC (eBio17B7) ThermoFisher Scientific Cat# 17-7177-81 anti-IFN-g Ab-PE (XMG1.2) ThermoFisher Scientific Cat# 12-7311-82 anti-GM-CSF Ab-FITC (MP1-22E9) ThermoFisher Scientific Cat# 11-7331-82 anti-Foxp3 Ab-APC (FJK-16s) ThermoFisher Scientific Cat# 17-5773-82 anti-RORgT Ab-PE (AFKJS-9) ThermoFisher Scientific Cat# 12-6988-82 anti-CD4 Ab-PerCP (RM4-5) ThermoFisher Scientific Cat# 46-0042-82 anti-CD8b Ab-PE (H35-17.2) ThermoFisher Scientific Cat# 12-0083-82 anti-CD44 Ab-PerCP-Cy5.5 (IM7) ThermoFisher Scientific Cat# 45-0441-80 anti-CD62L Ab-APC (MEL-14) ThermoFisher Scientific Cat# 17-0621-81 anti-CD25 Ab-APC (PC61.5) ThermoFisher Scientific Cat# 17-0251-81 anti-CD69 Ab-PerCP-Cy5.5 (H1.2F3) ThermoFisher Scientific Cat# 45-0691-80 anti-CCL3 Ab-eFluor660 (DNT3CC) Invitrogen Cat# 50-7532-82 anti-CCL5 Ab-APC (21445) Invitrogen Cat# MA5-23557 anti-mouse H3K4ac Ab-FITC (EPR16596) Abcam Cat# ab176799 anti-mouse H3K9ac Ab-FITC (C5B11) Cell Signaling Cat# 9649 anti-mouse histone H3 Ab-PE (D1H2) Cell Signaling Cat# 82241S anti-mouse H3K27ac Ab- FITC (D5E4) Cell Signaling Cat# 15485S anti-mouse H3K27me3 Ab (C36B11) Cell Signaling Cat# 9733 anti-mouse H3K4me3 Ab-PE (D1A9) Cell Signaling Cat# 55800S OKT3 BD Cat# 555330 anti-hCD28 antibody Bio X Cell Cat# BE0248 Goat anti-hamster antibody MP Biomedicals Cat# 0855397 anti-Acetyl-Histone H3 (Lys9) antibody (C5B11) Cell Signaling Cat# 9649 Anti-Glucose Transporter GLUT1 antibody Abcam Cat# ab115730 Bacterial and virus strains E. coli DH5a Thermo Fisher Scientific Cat# 18265017 (Continued on next page) Cell Reports 44, 115430, March 25, 2025 15

Techniques: Control, Cell Culture, Expressing, Injection, RNA Sequencing, Generated, Two Tailed Test

Journal: Cell reports

Article Title: Mitochondrial reactive oxygen species regulate acetyl-CoA flux between cytokine production and fatty acid synthesis in effector T cells.

doi: 10.1016/j.celrep.2025.115430

Figure Lengend Snippet: Figure 4. TMEM11 deficiency reduces Th1 effector cytokine production by increasing mitochondrial reactive oxygen species (A) Measurements of basal and oligomycin-induced maximal mitochondrial inner membrane potential in WT and Tmem11/ cells cultured under Th1-polarizing conditions and restimulated with anti-CD3 and anti-CD28 antibodies for indicated times. Bar graphs on the right show DMFI for tetramethylrhodamine-ethyl ester (TMRE) as calculated by subtracting the MFI values after carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP) treatment. Light gray flow plots show T cells with FCCP treatment. (B) Intracellular ATP levels in WT and Tmem11/ cells cultured under Th1-polarizing conditions. (C) Measurement of ROS levels in WT and Tmem11/ cells cultured under Th1-polarizing conditions after restimulation with anti-CD3 and anti-CD28 antibodies for the indicated times. (D) Cellular oxidative stress levels in WT and Tmem11/ cells cultured under Th1-polarizing conditions as determined by measuring the GSH/GSSH ratio after 2 h of restimulation with anti-CD3 and anti-CD28 antibodies. (E) Representative flow plots (left) and bar graphs (right) showing measurement of mtROS levels in WT and Tmem11/ cells cultured under Th1-polarizing conditions and treated overnight with 250 mM NAC and restimulated for indicated times in the presence of NAC. (F) Intracellular IFN-g measurement in WT and Tmem11/ cells cultured under Th1-polarizing conditions, treated overnight with 250 mM NAC, and restimulated with anti-CD3 and anti-CD28 antibodies for 5 h in the presence of NAC. Bar graphs in (A)–(F) show means ± SEM from three independent experiments; significance was determined by unpaired two-tailed t test. *p < 0.05, **p < 0.005, and ***p < 0.0005.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies TMEM11 Proteintech Cat# 16564-1-AP MIC60 Proteintech Cat# 10179-1-AP MIC10 Bioss antibodies Cat# BS-15029R OXPHOS Abcam Cat# MS604-300 OPA1 BD biosciences Cat# 612606 FLAG tag Millipore Sigma Cat# F3040 b-actin Santa Cruz Biotechnology Cat# sc-47778 anti-CD3 antibody Bio X Cell Cat# BE0001 anti-CD28 antibody Bio X Cell Cat# BE0015 anti-mIFN-g antibody Bio X Cell Cat# BE0055 anti-mIL-4 antibody Bio X Cell Cat# BE0045 anti-hIFN-g Ab-PE (45.B3) ThermoFisher Scientific Cat# 50-111-86 anti-IL-2 Ab-PE (MQ1-17H12) ThermoFisher Scientific Cat# 12-7029-81 anti-TNF Ab-APC (MAb11) ThermoFisher Scientific Cat# 17-7349-41 anti-T-bet Ab-APC (4B10) ThermoFisher Scientific Cat# 2124193 anti-IL-4 Ab-APC (11B11) ThermoFisher Scientific Cat# 17-7041-82 anti-IL-17A Ab-APC (eBio17B7) ThermoFisher Scientific Cat# 17-7177-81 anti-IFN-g Ab-PE (XMG1.2) ThermoFisher Scientific Cat# 12-7311-82 anti-GM-CSF Ab-FITC (MP1-22E9) ThermoFisher Scientific Cat# 11-7331-82 anti-Foxp3 Ab-APC (FJK-16s) ThermoFisher Scientific Cat# 17-5773-82 anti-RORgT Ab-PE (AFKJS-9) ThermoFisher Scientific Cat# 12-6988-82 anti-CD4 Ab-PerCP (RM4-5) ThermoFisher Scientific Cat# 46-0042-82 anti-CD8b Ab-PE (H35-17.2) ThermoFisher Scientific Cat# 12-0083-82 anti-CD44 Ab-PerCP-Cy5.5 (IM7) ThermoFisher Scientific Cat# 45-0441-80 anti-CD62L Ab-APC (MEL-14) ThermoFisher Scientific Cat# 17-0621-81 anti-CD25 Ab-APC (PC61.5) ThermoFisher Scientific Cat# 17-0251-81 anti-CD69 Ab-PerCP-Cy5.5 (H1.2F3) ThermoFisher Scientific Cat# 45-0691-80 anti-CCL3 Ab-eFluor660 (DNT3CC) Invitrogen Cat# 50-7532-82 anti-CCL5 Ab-APC (21445) Invitrogen Cat# MA5-23557 anti-mouse H3K4ac Ab-FITC (EPR16596) Abcam Cat# ab176799 anti-mouse H3K9ac Ab-FITC (C5B11) Cell Signaling Cat# 9649 anti-mouse histone H3 Ab-PE (D1H2) Cell Signaling Cat# 82241S anti-mouse H3K27ac Ab- FITC (D5E4) Cell Signaling Cat# 15485S anti-mouse H3K27me3 Ab (C36B11) Cell Signaling Cat# 9733 anti-mouse H3K4me3 Ab-PE (D1A9) Cell Signaling Cat# 55800S OKT3 BD Cat# 555330 anti-hCD28 antibody Bio X Cell Cat# BE0248 Goat anti-hamster antibody MP Biomedicals Cat# 0855397 anti-Acetyl-Histone H3 (Lys9) antibody (C5B11) Cell Signaling Cat# 9649 Anti-Glucose Transporter GLUT1 antibody Abcam Cat# ab115730 Bacterial and virus strains E. coli DH5a Thermo Fisher Scientific Cat# 18265017 (Continued on next page) Cell Reports 44, 115430, March 25, 2025 15

Techniques: Membrane, Cell Culture, Two Tailed Test

Journal: Cell reports

Article Title: Mitochondrial reactive oxygen species regulate acetyl-CoA flux between cytokine production and fatty acid synthesis in effector T cells.

doi: 10.1016/j.celrep.2025.115430

Figure Lengend Snippet: Figure 5. TMEM11 deficiency decreases H3K9Ac levels and increases fatty acid synthesis (A) Measurement of intracellular cytokine levels in WT and Tmem11/ cells cultured under Th1-polarizing conditions and treated with 20 mM sodium acetate (NaOAc) overnight and during restimulation with anti-CD3 and anti-CD28 antibodies for 5 h. (B) Measurement of H3K9 acetylation (H3K9Ac) levels in WT and Tmem11/ cells cultured under Th1-polarizing conditions. Cells were treated with 20 mM sodium acetate or 250 mM NAC overnight and during restimulation with anti-CD3 and anti-CD28 antibodies for 5 h. (C) Measurement of H3K9Ac levels of indicated gene loci in WT and Tmem11/ cells cultured under Th1-polarizing conditions using chromatin immunopre- cipitation. Cells were restimulated with anti-CD3 and anti-CD28 antibodies for 5 h before nucleus collection. (D) Whole-cell, nucleocytoplasmic, and mitochondrial levels of citrate and acetyl-CoA in WT and Tmem11/ cells cultured under Th1-polarizing conditions. Th1 cells were restimulated with anti-CD3 and anti-CD28 antibodies for 5 h. (E) Representative flow plots (left) and bar graph showing measurement of neutral lipid levels in WT and Tmem11/ cells cultured under Th1-polarizing conditions and restimulated for the indicated times.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies TMEM11 Proteintech Cat# 16564-1-AP MIC60 Proteintech Cat# 10179-1-AP MIC10 Bioss antibodies Cat# BS-15029R OXPHOS Abcam Cat# MS604-300 OPA1 BD biosciences Cat# 612606 FLAG tag Millipore Sigma Cat# F3040 b-actin Santa Cruz Biotechnology Cat# sc-47778 anti-CD3 antibody Bio X Cell Cat# BE0001 anti-CD28 antibody Bio X Cell Cat# BE0015 anti-mIFN-g antibody Bio X Cell Cat# BE0055 anti-mIL-4 antibody Bio X Cell Cat# BE0045 anti-hIFN-g Ab-PE (45.B3) ThermoFisher Scientific Cat# 50-111-86 anti-IL-2 Ab-PE (MQ1-17H12) ThermoFisher Scientific Cat# 12-7029-81 anti-TNF Ab-APC (MAb11) ThermoFisher Scientific Cat# 17-7349-41 anti-T-bet Ab-APC (4B10) ThermoFisher Scientific Cat# 2124193 anti-IL-4 Ab-APC (11B11) ThermoFisher Scientific Cat# 17-7041-82 anti-IL-17A Ab-APC (eBio17B7) ThermoFisher Scientific Cat# 17-7177-81 anti-IFN-g Ab-PE (XMG1.2) ThermoFisher Scientific Cat# 12-7311-82 anti-GM-CSF Ab-FITC (MP1-22E9) ThermoFisher Scientific Cat# 11-7331-82 anti-Foxp3 Ab-APC (FJK-16s) ThermoFisher Scientific Cat# 17-5773-82 anti-RORgT Ab-PE (AFKJS-9) ThermoFisher Scientific Cat# 12-6988-82 anti-CD4 Ab-PerCP (RM4-5) ThermoFisher Scientific Cat# 46-0042-82 anti-CD8b Ab-PE (H35-17.2) ThermoFisher Scientific Cat# 12-0083-82 anti-CD44 Ab-PerCP-Cy5.5 (IM7) ThermoFisher Scientific Cat# 45-0441-80 anti-CD62L Ab-APC (MEL-14) ThermoFisher Scientific Cat# 17-0621-81 anti-CD25 Ab-APC (PC61.5) ThermoFisher Scientific Cat# 17-0251-81 anti-CD69 Ab-PerCP-Cy5.5 (H1.2F3) ThermoFisher Scientific Cat# 45-0691-80 anti-CCL3 Ab-eFluor660 (DNT3CC) Invitrogen Cat# 50-7532-82 anti-CCL5 Ab-APC (21445) Invitrogen Cat# MA5-23557 anti-mouse H3K4ac Ab-FITC (EPR16596) Abcam Cat# ab176799 anti-mouse H3K9ac Ab-FITC (C5B11) Cell Signaling Cat# 9649 anti-mouse histone H3 Ab-PE (D1H2) Cell Signaling Cat# 82241S anti-mouse H3K27ac Ab- FITC (D5E4) Cell Signaling Cat# 15485S anti-mouse H3K27me3 Ab (C36B11) Cell Signaling Cat# 9733 anti-mouse H3K4me3 Ab-PE (D1A9) Cell Signaling Cat# 55800S OKT3 BD Cat# 555330 anti-hCD28 antibody Bio X Cell Cat# BE0248 Goat anti-hamster antibody MP Biomedicals Cat# 0855397 anti-Acetyl-Histone H3 (Lys9) antibody (C5B11) Cell Signaling Cat# 9649 Anti-Glucose Transporter GLUT1 antibody Abcam Cat# ab115730 Bacterial and virus strains E. coli DH5a Thermo Fisher Scientific Cat# 18265017 (Continued on next page) Cell Reports 44, 115430, March 25, 2025 15

Techniques: Cell Culture

Journal: Cell reports

Article Title: Mitochondrial reactive oxygen species regulate acetyl-CoA flux between cytokine production and fatty acid synthesis in effector T cells.

doi: 10.1016/j.celrep.2025.115430

Figure Lengend Snippet: Figure 6. Excessive ROS are sufficient to decrease cellular H3K9 acetylation and increase fatty acid synthesis in Th1 cells (A–D) Measurement of ROS (A), intracellular cytokines (B), cellular H3K9Ac (C), and neutral lipids (D) in WT T cells cultured under Th1-polarizing conditions after treating cells with indicated concentrations of extracellular H2O2 during restimulation with anti-CD3 and anti-CD28 antibodies for 5 h. Tmem11/ Th1 cells without H2O2 treatment were used for comparison of phenotypes. (E–H) Measurement of ROS (E), intracellular cytokines (F), cellular H3K9Ac (G), and neutral lipids (H) in WT T cells cultured under Th1-polarizing conditions after inhibition of ETC complex I using 10 mM rotenone during restimulation for 5 h. Inhibition of ETC complex II using 100 mM TTFA was used as a negative control for ROS generation. Tmem11/ Th1 cells without rotenone treatment were used for comparison of phenotypes. Individual points in bar graphs in (A)–(H) show technical replicates from two independent experiments. Data are shown as means ± SEM. Significance was determined by unpaired two-tailed t test. **p < 0.005 and ***p < 0.005.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies TMEM11 Proteintech Cat# 16564-1-AP MIC60 Proteintech Cat# 10179-1-AP MIC10 Bioss antibodies Cat# BS-15029R OXPHOS Abcam Cat# MS604-300 OPA1 BD biosciences Cat# 612606 FLAG tag Millipore Sigma Cat# F3040 b-actin Santa Cruz Biotechnology Cat# sc-47778 anti-CD3 antibody Bio X Cell Cat# BE0001 anti-CD28 antibody Bio X Cell Cat# BE0015 anti-mIFN-g antibody Bio X Cell Cat# BE0055 anti-mIL-4 antibody Bio X Cell Cat# BE0045 anti-hIFN-g Ab-PE (45.B3) ThermoFisher Scientific Cat# 50-111-86 anti-IL-2 Ab-PE (MQ1-17H12) ThermoFisher Scientific Cat# 12-7029-81 anti-TNF Ab-APC (MAb11) ThermoFisher Scientific Cat# 17-7349-41 anti-T-bet Ab-APC (4B10) ThermoFisher Scientific Cat# 2124193 anti-IL-4 Ab-APC (11B11) ThermoFisher Scientific Cat# 17-7041-82 anti-IL-17A Ab-APC (eBio17B7) ThermoFisher Scientific Cat# 17-7177-81 anti-IFN-g Ab-PE (XMG1.2) ThermoFisher Scientific Cat# 12-7311-82 anti-GM-CSF Ab-FITC (MP1-22E9) ThermoFisher Scientific Cat# 11-7331-82 anti-Foxp3 Ab-APC (FJK-16s) ThermoFisher Scientific Cat# 17-5773-82 anti-RORgT Ab-PE (AFKJS-9) ThermoFisher Scientific Cat# 12-6988-82 anti-CD4 Ab-PerCP (RM4-5) ThermoFisher Scientific Cat# 46-0042-82 anti-CD8b Ab-PE (H35-17.2) ThermoFisher Scientific Cat# 12-0083-82 anti-CD44 Ab-PerCP-Cy5.5 (IM7) ThermoFisher Scientific Cat# 45-0441-80 anti-CD62L Ab-APC (MEL-14) ThermoFisher Scientific Cat# 17-0621-81 anti-CD25 Ab-APC (PC61.5) ThermoFisher Scientific Cat# 17-0251-81 anti-CD69 Ab-PerCP-Cy5.5 (H1.2F3) ThermoFisher Scientific Cat# 45-0691-80 anti-CCL3 Ab-eFluor660 (DNT3CC) Invitrogen Cat# 50-7532-82 anti-CCL5 Ab-APC (21445) Invitrogen Cat# MA5-23557 anti-mouse H3K4ac Ab-FITC (EPR16596) Abcam Cat# ab176799 anti-mouse H3K9ac Ab-FITC (C5B11) Cell Signaling Cat# 9649 anti-mouse histone H3 Ab-PE (D1H2) Cell Signaling Cat# 82241S anti-mouse H3K27ac Ab- FITC (D5E4) Cell Signaling Cat# 15485S anti-mouse H3K27me3 Ab (C36B11) Cell Signaling Cat# 9733 anti-mouse H3K4me3 Ab-PE (D1A9) Cell Signaling Cat# 55800S OKT3 BD Cat# 555330 anti-hCD28 antibody Bio X Cell Cat# BE0248 Goat anti-hamster antibody MP Biomedicals Cat# 0855397 anti-Acetyl-Histone H3 (Lys9) antibody (C5B11) Cell Signaling Cat# 9649 Anti-Glucose Transporter GLUT1 antibody Abcam Cat# ab115730 Bacterial and virus strains E. coli DH5a Thermo Fisher Scientific Cat# 18265017 (Continued on next page) Cell Reports 44, 115430, March 25, 2025 15

Techniques: Cell Culture, Comparison, Inhibition, Negative Control, Two Tailed Test